Bottom Line:
Here we use components of the Mesoplasma florum transfer-messenger RNA system to create a synthetic degradation system that provides both independent control of steady-state protein level and inducible degradation of targeted proteins in Escherichia coli.We demonstrate application of this system in synthetic circuit development and control of core bacterial processes and antibacterial targets, and we transfer the system to Lactococcus lactis to establish its broad functionality in bacteria.We create a 238-member library of tagged essential proteins in E. coli that can serve as both a research tool to study essential gene function and an applied system for antibiotic discovery.

ABSTRACTTunable control of protein degradation in bacteria would provide a powerful research tool. Here we use components of the Mesoplasma florum transfer-messenger RNA system to create a synthetic degradation system that provides both independent control of steady-state protein level and inducible degradation of targeted proteins in Escherichia coli. We demonstrate application of this system in synthetic circuit development and control of core bacterial processes and antibacterial targets, and we transfer the system to Lactococcus lactis to establish its broad functionality in bacteria. We create a 238-member library of tagged essential proteins in E. coli that can serve as both a research tool to study essential gene function and an applied system for antibiotic discovery. Our synthetic protein degradation system is modular, does not require disruption of host systems and can be transferred to diverse bacteria with minimal modification.

Figure 3: Protease-driven control of a synthetic toggle switch(a) Schematic of the synthetic toggle switch in which reciprocal transcriptional repression by TetR and LacI form a bistable circuit. GFP and mCherry serve as fluorescent reporters for the LacI+ and TetR+ toggle states, respectively. Addition of a pdt tag to LacI enables a protease-driven switch from the GFP+ to the mCherry+ state. (b) Flow cytometry scatter plots show GFP and mCherry fluorescence 0, 4 and 8 hours after mf-Lon expression from the inducible promoter PBAD. Degradation of LacI-pdt#3 causes the toggle to switch from the GFP+ state to the mCherry+ state by 8 hours, while the untagged toggle remains in the GFP+ state. Magenta lines indicate the gate parameters used to define the GFP+ and mCherry+ states: cells bounded in the lower left quadrant are considered negative for both GFP and mCherry. (c) The percentage of cells in the mCherry+ state following mf-Lon induction with 1 mM arabinose. Data collected by flow cytometry were measured using the parameters shown in (b) and represent the mean of three biological replicates. For all LacI-pdt variants, P < 0.001 when compared to untagged LacI at 24 hours after induction. Error bars show the standard deviation. See Supplementary Figure 6 for data showing that non-induced strains did not shift to mCherry+.

Mentions:
To demonstrate the use of this system to control engineered genetic circuits, we used pdt fusions to provide post-translational control of a transcription-based toggle switch12. As shown in Figure 3a, LacI and TetR were previously engineered to form a bistable circuit based on reciprocal repression, and concomitant regulation of GFP and mCherry allows facile fluorescence-based identification of the toggle switch state13. We fused pdt#3 to the C-terminus of LacI in the toggle circuit and used the arabinose-inducible PBAD promoter1 to drive mf-Lon expression from a second plasmid. Upon mf-Lon induction, the circuit containing LacI-pdt#3 switched from the LacI+/GFP+ state to the TetR+/mCherry+ state within 8 hours of mf-Lon induction, while the untagged circuit remained unchanged (Fig. 3b). Moreover, substitution of LacI-pdt#3 with the hybrid tags pdt#3A and pdt#3B provided temporal control over the circuit switch rate (Fig. 3c), and pdt fusions to TetR enabled mf-Lon to switch the toggle in the opposite direction (Supplementary Fig. 5). Importantly, the LacI-pdt circuits maintained transcription-based bistability in the absence of mf-Lon induction, demonstrating the ability of the system to leave existing regulatory networks intact (Supplementary Fig. 6).

Figure 3: Protease-driven control of a synthetic toggle switch(a) Schematic of the synthetic toggle switch in which reciprocal transcriptional repression by TetR and LacI form a bistable circuit. GFP and mCherry serve as fluorescent reporters for the LacI+ and TetR+ toggle states, respectively. Addition of a pdt tag to LacI enables a protease-driven switch from the GFP+ to the mCherry+ state. (b) Flow cytometry scatter plots show GFP and mCherry fluorescence 0, 4 and 8 hours after mf-Lon expression from the inducible promoter PBAD. Degradation of LacI-pdt#3 causes the toggle to switch from the GFP+ state to the mCherry+ state by 8 hours, while the untagged toggle remains in the GFP+ state. Magenta lines indicate the gate parameters used to define the GFP+ and mCherry+ states: cells bounded in the lower left quadrant are considered negative for both GFP and mCherry. (c) The percentage of cells in the mCherry+ state following mf-Lon induction with 1 mM arabinose. Data collected by flow cytometry were measured using the parameters shown in (b) and represent the mean of three biological replicates. For all LacI-pdt variants, P < 0.001 when compared to untagged LacI at 24 hours after induction. Error bars show the standard deviation. See Supplementary Figure 6 for data showing that non-induced strains did not shift to mCherry+.

Mentions:
To demonstrate the use of this system to control engineered genetic circuits, we used pdt fusions to provide post-translational control of a transcription-based toggle switch12. As shown in Figure 3a, LacI and TetR were previously engineered to form a bistable circuit based on reciprocal repression, and concomitant regulation of GFP and mCherry allows facile fluorescence-based identification of the toggle switch state13. We fused pdt#3 to the C-terminus of LacI in the toggle circuit and used the arabinose-inducible PBAD promoter1 to drive mf-Lon expression from a second plasmid. Upon mf-Lon induction, the circuit containing LacI-pdt#3 switched from the LacI+/GFP+ state to the TetR+/mCherry+ state within 8 hours of mf-Lon induction, while the untagged circuit remained unchanged (Fig. 3b). Moreover, substitution of LacI-pdt#3 with the hybrid tags pdt#3A and pdt#3B provided temporal control over the circuit switch rate (Fig. 3c), and pdt fusions to TetR enabled mf-Lon to switch the toggle in the opposite direction (Supplementary Fig. 5). Importantly, the LacI-pdt circuits maintained transcription-based bistability in the absence of mf-Lon induction, demonstrating the ability of the system to leave existing regulatory networks intact (Supplementary Fig. 6).

Bottom Line:
Here we use components of the Mesoplasma florum transfer-messenger RNA system to create a synthetic degradation system that provides both independent control of steady-state protein level and inducible degradation of targeted proteins in Escherichia coli.We demonstrate application of this system in synthetic circuit development and control of core bacterial processes and antibacterial targets, and we transfer the system to Lactococcus lactis to establish its broad functionality in bacteria.We create a 238-member library of tagged essential proteins in E. coli that can serve as both a research tool to study essential gene function and an applied system for antibiotic discovery.

ABSTRACTTunable control of protein degradation in bacteria would provide a powerful research tool. Here we use components of the Mesoplasma florum transfer-messenger RNA system to create a synthetic degradation system that provides both independent control of steady-state protein level and inducible degradation of targeted proteins in Escherichia coli. We demonstrate application of this system in synthetic circuit development and control of core bacterial processes and antibacterial targets, and we transfer the system to Lactococcus lactis to establish its broad functionality in bacteria. We create a 238-member library of tagged essential proteins in E. coli that can serve as both a research tool to study essential gene function and an applied system for antibiotic discovery. Our synthetic protein degradation system is modular, does not require disruption of host systems and can be transferred to diverse bacteria with minimal modification.